FR2878862A1 - POLYNUCLEOTIDE SEQUENCES WITH SPECIFIC PROMOTER ACTIVITY OF PLANT ROOT CELLS - Google Patents

Abstract

The present invention relates to isolated polynucleotides with promoter activity, allowing the specific expression of heterologous sequences of interest in the cells of the roots of plants, depending on the cell base and the desired stage of development, throughout. the growth of the plant, as well as the transgenic plants comprising them.

Description

The present invention relates to isolated polynucleotides with activity

  promoter allowing the specific expression of heterologous sequences of interest in the cells of plant roots as a function of

  the cell base and the desired stage of development throughout the growth of the plant as well as the transgenic plants comprising them.

  The control of the enemies of the cultures is one of the keys which condition the global food balance. Chemistry has long provided the bulk of the effort in the fight against pests. Recently, other methods have also been used, such as biological control and the creation of resistant varieties, for which transgenesis is a tool of choice.

  Many plant pathogens of viral, bacterial or phytoplasmic origin are not sensitive to currently marketed pesticides. The only solution is to use resistant plant varieties. In addition, certain pathogenic fungi, mites, nematodes or insects can withstand high doses of pesticides without damage.

  Transgenesis broadens this investigation potential by allowing, on the one hand, the exploitation of genes from other varieties, other species or even other genera, on the other hand, by quantitative control (modulate the intensity of gene expression) and qualitative (expressing the gene in this part of the plant) of their expression. The first results, notably the creation of varieties resistant to viral diseases (resistance to short-knot in the vine), to nematodes or to pests (maize resistant to the moth) are particularly promising.

  Now, for a large number of applications, it is not necessary that the protein of interest conferring the desired agronomic property has a distributed expression in all the organs and / or cell types of the transformed plant.

  Very early, the search for a more specific expression of the gene of interest was undertaken and led, for example, to the identification of specific promoters of tissue or organ.

  In particular, a promoter directing the expression of a polynucleotide of interest in a manner both strong and targeted in the root would allow many applications that can be defined both in the defense against pathogens such as bacteria , fungi, nematodes or insects, resistance to stress (cold, water, salt stress), quality improvement (for example: increase the sucrose content in sugar beet), nutrition (example: express a nitrate transporter gene).

  There are many applications in the prior art for promoters to direct expression in plant roots. For example, Canadian Patent Application CA2425886 entitled Rootspecific conifer gene promoter and its use relates to a root-promoting polynucleotide sequence derived from the pine PR10 gene. Canadian Patent Application CA2228046 entitled: Root Cortex specific gene promoter refers to an isolated DNA comprising a promoter sequence which directs the transcription of a heterologous DNA segment specifically into the root cortex of a plant cell, an example of transformation in the tobacco plant. The application WO 03/040322 entitled Promoters regulating gene expression in plant roots relates to a promoter isolated from maize and its functional equivalents which has the particularity of leading an expression of heterologous genes specifically in the roots in order to increase the characteristics. agronomic, horticultural and / or pesticidal plant and plant tissues comprising the promoters according to the invention; WO 02/46439 entitled: New root-specific promoters activating the expression of a novel LLR domain receptor kinase discloses nucleic acids encoding root-specific promoter transcriptional regulators and nucleic acids encoding a new LLR receptor protein receptor, US Pat. No. 5,633,363 Root preferential promoter refers to promoter fragments isolated from maize and more particularly from an upstream region of 4.7Kb designated ZRP2 as well as its functional equivalents which have the particularity of conducting an expression heterologous genes specifically in the roots to increase the agronomic, horticultural and / or pesticidal characteristics of a plant or the US application 5,401,836 entitled: Brassica regulatory sequence for root-specific or root-abundant gene expression concerning the isolation of a promoter that shows a strong expression in Brassica sp. This promoter is useful for conducting heterologous protein transcription which confers immunity or resistance to pathologies likely to affect the roots and in particular infection by fungi, bacteria and insects.

  None of these Requests allows to obtain an expression of sequences of interest according to the needs and especially by choosing the stage of the development and targeting precisely the cellular base where one wants to direct the expression.

  The sequences according to the invention make it possible to obtain a specific expression of genes of interest at all stages of the development of the plant, for example an expression of antibiotic or proteins with herbicidal activity specifically in roots at a very early stage. to avoid their traces in the adult stage and also allowing a very localized expression according to the cell base which can have an importance according to the type of agriculture.

  The present invention. therefore relates to a method for directing the expression of a nucleotide sequence of interest in cells of the root of a plant characterized in that an isolated nucleic acid comprising a plant promoter selected from the following sequences is used .

  EAG 96 (SEQ ID NO: 1), AAJ 3 (SEQ ID NO: 2), DRM 33 (SEQ ID NO: 3), DUA 2 (SEQ ID NO: 4), DSM 153 (SEQ ID NO: 5), DYK 138 (SEQ ID NO. N 6), DXP 4 (SEQ ID NO: 7), EAD 29 (SEQ ID NO: 8), DYC 237 (SEQ ID NO: 9), DUR17 (SEQ ID NO: 10), FAE 39 (SEQ ID NO: 11) and DRW 2 ( SEQ ID N 12) Preferably, a nucleic acid according to the invention is in isolated or purified form.

  The term "isolated" in the sense of the present invention refers to biological material that has been removed from its original environment (the environment in which it is naturally located). For example, a polynucleotide naturally occurring in a plant or animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the plant or animal is isolated.

  Such a polynucleotide may be included in a vector and / or in a composition and still remain in the isolated state because the vector or composition does not constitute its natural environment.

  The term purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds.

  For purposes of the present description, the term nucleotide sequence may be used to denote either a polynucleotide or a nucleic acid. The term nucleotide sequence includes the genetic material itself and is therefore not restricted to information concerning its sequence.

  The invention also relates to a method, characterized in that the sequence is chosen from a polynucleotide having at least 80% nucleotide identity with a polynucleotide according to the invention or a nucleic acid of sequence complementary thereto. .

  According to the present invention,% identity is understood to mean the percentage of identical nucleotides that can be calculated by those skilled in the art using a computer program for sequence comparison such as Blast (www.ncbi.nlm.nih.gov). and the program FastDB with the following parameters: Mistmatch penalty: 1.00; Gap penalty: 1.00; Gap Size Penalty: 0.33; joining penalty: 30.0. These algorithms are presented in Current Methods in Sequencing and Synthesis Methods and Applications, pp. 127-149, 1988, Ala. R. Liss, Inc., incorporated in the

description by reference.

  Polynucleotides according to the invention can also be defined by their selective hybridization properties with one of the polynucleotides defined by the sequences SEQ ID No. 1 to SEQ ID No. 12 under conditions of high stringency as defined in Sambrook et al. Molecular Cloning A Laboratory Manual (Cold Spring Harbor Press, 1989) at paragraphs 11.1 to 11.61.

  The nucleotide differences that can comprise a nucleic acid according to the invention with respect to the nucleotide sequences SEQ ID N 1 to SEQ ID N 12 can result in substitutions, deletions or additions of one or more consecutive nucleotides or not.

  Also included in the invention are nucleic acids comprising all or part of a polynucleotide having at least 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.8% identity. in nucleotides with the nucleotide sequences SEQ ID N 1 to SEQ ID N 12, or a nucleic acid of complementary sequence.

  According to another aspect, the invention also relates to a nucleic acid characterized in that it comprises all or part of a hybridizing polynucleotide, under conditions of high stringency, with the sequences SEQ ID N 1 to SEQ ID N 12 according to the invention, or a nucleic acid of complementary sequence.

  In general, conditions of high stringency, for a given polynucleotide can be obtained by operating at a temperature of 5 to 10 C below the melting point of the duplex consisting of said polynucleotide and its complement, in a medium of a given composition. Example of high stringency hybridization conditions usable in a large number of cases, indicate the following conditions: Prehybridization: Same conditions as for hybridization Duration: 1 night.

  Hybridization: 5X SSPE (0.9 M NaCl, 50 mM sodium phosphate pH 7.7, 5 mM EDTA) 5X Denhardt's (0.2% PVP, 0.2% Ficoll, 0.2% BSA) 0.1% SDS Duration: 1 night.

  Washings: 2X SSC, 0.1% SDS 10 min 65 C 1X SSC, 0.1% SDS 10 min 65 C 0.5X SSC, 0.1% SDS 10 min 65 C 0.1X SSC, 0.1% SDS 10 min 65 C By part of a promoter polynucleotide according to the invention is meant a nucleotide sequence of a base length smaller than that of the sequence according to the invention and retaining the ability to direct the expression of a sequence nucleotide of interest in the cells of the root of a plant.

  A part of a promoter polynucleotide according to the invention can also be obtained for example by deletion of one or more nucleotides of the polynucleotide of sequence SEQ ID No. 1 to SEQ ID No. 12 using the exonuclease III techniques (Ausubel et al. al., 1989). A polynucleotide part of the plant promoter according to the invention advantageously has a length in nucleotide ranging from 200, 250, 300, 400, 500, 750, 1000, 1200, 1500 or 2000 nucleotides (or base pairs if it is under the double-stranded form).

  For the implementation of restriction enzymes to obtain polynucleotide fragments corresponding to a portion of a polynucleotide promoter according to the invention, the skilled person can advantageously refer to the work of Sambrook et al. . (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

  A promoter polynucleotide portion according to the invention may also be prepared by specific amplification of the fragment of interest using a pair of primers flanking, respectively on the 5 'side and the 3' side, the sequence of interest. , for example using the PCR method, as described in particular in US Patents 4,683,195, US 4,683,202 and US 4,965,188.

  The biological activity of a part of a promoter polynucleotide according to the invention can easily be verified by those skilled in the art, in particular by means of vector constructs and methods of transforming plants with these, such as described in

examples.

  The invention also relates to a recombinant expression cassette characterized in that it comprises a promoter consisting of a polynucleotide sequence as defined above and a heterologous sequence of interest placed under transcriptional control of said promoter and whose expression is sought in the cells of the root of a plant.

  Advantageously, such a nucleic acid will comprise a nucleotide sequence of interest chosen from gene coding sequences interacting with parasites or pathogens such as nematodes or fungi, such as for example a polynucleotide encoding a protein with herbicidal activity or antibiotic or the coding sequences of glucanase, said nucleotide sequence of interest being placed under the control of a promoter polynucleotide according to the invention.

  It may also be sequences of endochitinases such as those described in European Patent 493,581 or else gene savers acting on the sugar content of the plant.

  By way of example, the coding sequences of genes of interest ensuring the protection of a plant against other stress conditions may advantageously be placed under the control of a polynucleotide promoter according to the invention, for water stress or saline: arskl gene (Hwang et al., 1995), cDNA pA9 (Winicov, I. Deutch SE 1994) or cDNA Alfin 1 (Bastola, DR et al., 1998).

  Other coding sequences could be used under the control of the promoters according to the invention, for example to act on the sucrose content of sugar beet: BvSPS1 gene (Hesse H. et al., 1995), or to overexpress a gene already expressed physiologically as NRT1 or NRT2 nitrate transporter genes (Crawford, NL et al 1998, Leah R. et al., 1991).

  The invention is furthermore related to nucleotide fragments comprising 10 to 2000 consecutive nucleotides of a nucleic acid according to the invention, in particular fragments having a promoter activity similar or identical to the promoter activity of the corresponding complete sequence, preferentially a nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1 to SEQ ID No. 12 or a hybridizing nucleic acid, under high stringency hybridization conditions with the nucleotide sequences according to the invention or a nucleic acid of complementary sequence.

  Preferably, such fragments will have a length of 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 or 2000 consecutive nucleotides of a promoter polynucleotide according to the invention or consisting of fragments of a length of 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 or 2000 consecutive nucleotides of a polynucleotide promoter according to the invention. Such nucleotide fragments may advantageously be used as probes or nucleotide primers for the purpose of detecting or amplifying all or part of a sequence with specific promoter activity of plant roots according to the invention.

  According to another aspect, the invention relates to a recombinant cloning and / or expression vector comprising a promoter polynucleotide according to the invention and more particularly comprising at least one expression cassette according to the invention. Such a recombinant vector will advantageously comprise a nucleotide sequence of interest placed under the control of said plant promoter. Vectors which may be used for the purposes of the present invention include the following: pBIN19 vector (Bevan et al., 1984, Nucleic Acids Research, Vol 12: 8711-8,721, commercially available from Clontech Corporation, Pale Alto, California, USA) ); vector 101 (Jefferson, 1987, PlantMolecular Biology Reporter, 5: 387-405, marketed by Clontech); pB1221 vector (Jefferson, 198, Plant Molecular Biology Reporter, vol.5: 387--405, commercially available from CLONTECH), pBI121 vector (Jefferson, 1987, Plant Molecular Biology Reporter, 5: 387-405, marketed by Clontech Corporation); pEGFP vector (Cormack, B.P. et al., 1996, Yang T.T. et al., 1996, marketed by the Clontech Company), or the vector pC-gus.

  The invention further relates to a host cell transformed with at least one expression cassette according to the invention or a recombinant vector as defined above. The invention particularly relates to a recombinant host cell, characterized in that it comprises a nucleic acid with plant promoter activity specific to plant roots according to the invention, optionally associated with a polynucleotide of interest placed under the control of the latter. , or a recombinant vector as defined above. The preferred recombinant host cells according to the invention can be indifferently of bacterial or plant origin. Thus, in particular, bacterial cells of different E. coli strains can be used. coli or Agrobacterium tumefaciens. They are also plant cells transformed with a vector according to the invention, such as Arabidopsis thaliana, rapeseed, tobacco or corn cells. The invention also relates to a recombinant plant multicellular organism characterized in that it comprises recombinant host cells as defined above.

  In a particular embodiment of the invention, this relates to a transgenic plant generated from a recombinant host cell according to the invention as well as to the tissues or parts of said plants and also all the transgenic plants or parts thereof. of a transgenic plant transformed with at least one expression cassette according to the invention.

  The term "plant tissue" refers to any tissue of a plant, plant or culture. This term includes whole plants, plant cells, plant organs, plant seeds, protoplasts, calli, cell cultures and any other plant cells organized as functional and / or structural unit. Parts of regenerated plants such as flowers, seeds, leaves, stems, fruits, pollen, tubers, wood and the like are also within the scope of the invention.

  A transgenic plant according to the invention may be in particular a rapeseed, a tobacco, a maize, wheat, barley, sunflower or Arabidopsis thaliana.

  The transgenic plants as defined above therefore have the property of expressing a nucleotide sequence of interest specifically at the different cell types of the root (from the outside to the inside: epidermis, cortex, endoderm, pericycle, vessel), at all stages of development of the plant.

  The subject of the invention is also a process for obtaining a transgenic plant expressing specifically a nucleotide sequence of interest in the cells of the root at all stages of development of said plant, characterized in that it comprises the steps following.

  a) Obtaining a plant recombinant host cell according to the invention; b) Regeneration of an entire plant from the recombinant host cell obtained in step a) c) Selection of plants obtained in step b) having integrated the nucleotide sequence of interest placed under the control of the plant polynucleotide promoter according to the invention.

  Optionally, the method according to the invention comprises a step of crossing transgenic plants with one another as obtained previously or else the crossing between a transgenic plant according to the invention and a plant of the same species and the selection of the plants resulting from the crossing. having preserved the transgene.

  The invention further relates to a process for obtaining a transgenic plant characterized in that it comprises the following steps: a) transformation of at least one plant cell with a vector according to the invention; b) culturing said transformed cells in order to generate a plant containing in its genome an expression cassette as defined according to the invention.

  The invention also relates to a transgenic plant as obtained by any of the above methods. Preferably, a transgenic plant according to the invention has not only integrated into its genome a transgene comprising a nucleotide sequence of interest placed under the control of the plant promoter polynucleotide presently described but expresses said nucleotide sequence of interest mainly or exclusively in the constituent cells of the root.

  Finally, the invention also relates to the seeds obtained from a transgenic plant according to the invention. It is in particular a seed of Arabidopsis thaliana, rapeseed, tobacco or maize having incorporated a nucleic acid according to the invention.

  The present invention also relates to a method for protecting a plant from infection by a parasite comprising the following steps: a) Transformation of the plant with an expression cassette according to any one of claims 3 to 4; b) Selection of the plant having integrated the expression cassette.

  Finally, the invention relates to the use of a promoter nucleotide sequence according to the invention for expressing a gene in root cells and, in a particular embodiment of the invention, for expressing a sequence of interest. with herbicidal or antibiotic activity.

  The invention will be further illustrated, without being limited, by the following figures and examples.

  EXAMPLE 1: PREPARATION OF TRANSFORMANTS The promoter sequences were amplified by PCR (polymerase chain reaction) reaction using the respective primer pairs described below (SEQ ID N 13 to SEQ ID N 36). The DNA (10 to 50 ng) from Arabidopsis thaliana ecotype Wassilewskija was used as a template.

  The reactions were carried out thanks to the Expand High Fidelity kit (Roche) according to the supplier's protocol. PCR was performed with 30 cycles (55 C 30s for pairing, 72 C elongation for 3 min and 94 C for 30s).

  The amplified DNA fragments were cloned into the pGEM-T Easy vector (Promega). After verification of the amplified sequences, the latter were cloned into the binary vector pBI 101 (Jefferson et al., 1987, EMBO J. 6, 3901-3907) using the restriction sites introduced into the oligonucleotides or from the pGEM polylinker. -T Easy, respectively: SmaI / BamHI (DRM33, AAJ3, DUA2, EAD 29); HindIII / BamHI (DYK 138, DSM 153, DYC 237, EAG 96) and SalI / BamHI (DUR 17, DXP 4, EAF 39, DRW 2). This cloning makes it possible to introduce the various promoters upstream of the GUS gene and of the NOS terminator into a binary vector. These vectors were then introduced by agro-transformation into Arabidopsis thaliana ecotype Wassilewskija plants for verification of their functionality by the vacuum infiltration technique (Bechtold et al, 1993, CR Acad Sci Paris 316, 1188-1193). ). The transformants were selected by virtue of the resistance to kanamycin (50 mg / L) carried by the vector pBi101. For each promoter, 16 plants per construct were transformed. The number of transformants obtained was quite variable (11 (DSM 153), 10 (DRM 33), 16 (DYK 138), 11 (DXP 4), 7 (EAD 29), 2 (DUA 2), 4 (AAJ 3), (DYC237), (DUR17), 3 (DRW2), 6 (EAF 39)). The staining patterns were reproducible between the lines, but could have a variable level of expression. Transformant analysis (expression of the GUS reporter gene) was performed by histochemical staining (Jefferson et al., 1987, EMBO J. 6, 3901-3907) at four distinct growth stages to study the expression profile of the promoter. throughout the growth of the plant (germination (stage 2 cotyledons), rosette stage (15 days after germination, adult plant (3-4 weeks) and staining of embryos.) The plants are derived from in vitro culture for selection antibiotics (Hoagland medium modified according to Sarrobert et al., 2000, Plant J. 24, 357-367), grown in culture chamber (16h day at 24 C and 8h night at 21 C, 150 pmol.m 2. s-1) to the rosette stage (15 days after germination), then the plants are subcultured in a growing chamber on the soil for the rest of the growth (16h day at 24 C and 8h night at 21 C, 300} m-2, s-1) EXAMPLE 2: VISUALIZATION OF SPECIFIC EXPRESSIONS FIGS. Sections of the transformed plants are described as described in Example 1, making it possible to visualize the colorations obtained at the different stages of growth. Several stages are indicated only in cases where the color changes or changes during development, especially young seedlings 8 to 14 days after sowing with details (cutting and / or enlargement) of the various areas of the root and the aerial part .

  The coloration observed is a 24h GUS coloration. These FIGS. 1 to 12 correspond to the colorations observed for the sequences SEQ ID No. 1 to SEQ ID No. 12 respectively.

  Figure 1: Expression profile of the EAG96 promoter visualized on 1 transformant.

  The specific expression is at the level of the epidermis, the cortex, and in the mature root. No discoloration is observed in the leaves, stems, pods, and embryo.

  Figure 2: Expression profile of the AAJ 3 promoter visualized on 4 transformants.

  Color is observed all over the root. It is very specific to the root except for the zone of the primary meristem. No coloration is observed in the aerial parts. It is a specific root promoter, constitutive.

  Figure 3: Expression profile of the DRM33 promoter visualized on 6 transformants.

  The expression GUS affects the embryo at a very early stage but not the reserves of the seed. The expression is visible in the root but not in the cap (part of the apex that touches the root). The expression is visible in the differentiated central part but not in the meristematic zone outside the epidermis. The expression is light in some transformants for the aerial part but it is not systematic.

  Leaves appear to be affected in conductive tissue throughout the plant. We find the conductive fabrics throughout the plant. The expression is mainly root. A staining is observed in the cotyledons that are not found in old leaves.

  Figure 4: Expression profile of the DUA2 promoter visualized on 2 transformants.

  Staining is observed in the embryo at the heart-shaped stage, but nothing in the flowers and the hypocotyl. It is a promoter with very strong expression in the root.

  Figure 5: Expression profile of the DSM153 promoter visualized on 11 transformants.

  A strong staining is observed in the Apex, which continues in the roots in the epidermis and in the aged parts. No expression is observed in the siliques or in the stems. There is a weak expression at the base of the flowers. Expression in young leaves is sometimes observed in some transformants but not in old leaves. It is a strong root promoter.

  Figure 6: Expression profile of the DYK138 promoter visualized on 13 transformants.

  Stomatal and hydathodic staining was observed. Staining is observed in the aged leaves, in the embryo, and in the siliques. Color is observed throughout the root.

  Figure 7: Expression profile of the DXP4 promoter visualized on 9 transformants.

  A visible coloration is observed throughout the seed in a very homogeneous manner. However, it can be noticed that the expression is very strong in the zone of division as well as in the leaf and root meristem.

  Color is observed all over the root except in the epidermis and the cortex.

  Figure 8: Expression profile of the EAD29 promoter visualized on 6 transformants.

  We observe an expression in the columella, the central cylinder, the embryo, on all the bases except the cortex and the epidermis. No discolouration was observed in the aerial parts, with the exception of cotyledon and leaves (stages 1 and 2). Slight expression is observed in hydathodes, and apical meristem in some transformants.

  Figure 9: Expression profile of the DYC237 promoter visualized on 2 transformants.

  GUS expression is observed in the epidermis, columellae, meristem, areas of elongation and differentiation, and in the root at all stages.

  Figure 10: Expression profile of the DUR17 promoter visualized on 6 transformants.

  Constitutive expression is observed in all courses at all stages except at the central cylinder.

  Figure 11: Expression profile of the EAF39 promoter visualized on 4 transformants.

  We observe a color of the xylème until the pericycle face and nowhere else. The color forms a fan. The signal diffuses through cells.

  Figure 12: Expression profile of the DRW2 promoter visualized on 2 transformants.

  Color is observed throughout the root in the young parts, and the central cylinder in the aged parts, as well as in the embryo.

  Results Table 1: Expression profiles of a gene of interest according to the promoter used and according to the cellular localization and the stage of development of the plant.

  (Stage 1) (Stage 2) (Stage 3) (Stage 4) Germination of young seedlings Plants in greenhouse Adult plants cotyledons rosette stage with fruits DSM153 DSM153 DSM153 DSM153 DYK138 DYK138 DYK138 DYK138 Meristem DXP4 DXP4 DXP4 DXP4 primary EAD29 EAD29 EAD29 EAD29 DUA2 dua2 dua2 dua2 AAJ3 AAJ3 AAJ3 AAJ3 DYC237 DYC237 DSM153 DSM153 DSM153 DSM153 DYK138 DYK138 DYK138 DYK138 meristem DXP4 DXP4 DXP4 DXP4 secondary EAD29 EAD29 EAD29 EAD29 dua2 dua2 dua2 dua2 AAJ3 AAJ3 AAJ3 AAJ3 DYC237 DYC237 DSM153 DSM153 DSM153 DSM153 DRM33 DRM33 DRM33 DRM33 DYK138 DYK138 DYK138 DYK138 DXP4 DXP4 DXP4 DXP4 Zone EAD29 EAD29 EAD29 EAD29 elongation EAG96 EAG96 dua2 dua2 dua2 dua2 DRw2 DRw2 DRw2 DRw2 DYC237 DYC237 DYC237 DYC237 EAF39 EAF39 DUR17 DUR17 DUR17 DUR17 DSM153 DSM153 DSM153 DSM153 DRM33 DRM33 DRM33 DRM33 Zone DYK138 DYK138 DYK138 DYK138 differentiation DXP4 DXP4 DXP4 DXP4 EAD29 EAD29 EAD29 EAD29 EAG96 EAG96 r- DU DUA2 DUA2 DUA2 DUA2 AAJ3 AAJ3 AAJ3 AAJ3 DRW2 DRW2 DRW2 DRW2 DYC237 DYC237 DYC237 DYC237 EAF39 EAF39 DUR17 DUR17 DUR17 DUR17 DSM153 DSM153 DSM153 DSM153 DRM33 DRM33 DRM33 DRM33 DYK138 DYK138 DYK138 DYK138 DXP4 DXP4 DXP4 DXP4 EAD29 EAD29 EAD29 EAD29 Racine EAG96 EAG96 EAG96 EAG96 Mature dua2 dua2 dua2 dua2 AAJ3 AAJ3 AAJ3 AAJ3 DRw2 DRw2 DRw2 DRw2 DYC237 DYC237 DYC237 DYC237 EAF39 EAF39 DUR17 DUR17 DUR17 DUR17 DSM153 DSM153 DRM33 DRM33 DYK138 DYK138 cotyledon EAD29 EAD29 dua2 dua2 DRw2 DRw2 Hypocotyl DXP4 DXP4 DSM153 DSM153 DSM153 DSM153 DRM33 DRM33 DYK138 DYK138 DYK138 DYK138 Leaves DXP4 DXP4 DXP4 DXP4 EAD29 EAD29 dua2 dua2 DRw2 DRw2 Flowers DXP4 DXP4 Stomate DYK138 DYK138 dua2 DUA2 DRM33 DRM33 DYK138 DYK138 Embryo DXP4 DXP4 EAD29 EAD29 DUA2 DUA2 promoters according to the invention makes it possible to target all the stages of the development of the plant as well as all the bases depending on the place where it is desired to express a gene of interest . The

  DRW2 DRW2 DUR17 results show that the DUR17 group

Claims (17)

  A method for directing the expression of a nucleotide sequence of interest in cells of the root of a plant, characterized in that an isolated polynucleotide comprising a plant promoter selected from the following sequences is used: a) a polynucleotide having at least 80% nucleotide identity with a polynucleotide selected from the following sequences: EAG 96 (SEQ ID N 1), AAJ 3 (SEQ ID N 2), DRM 33 (SEQ ID NO: 3), DUA 2 ( SEQ ID N 4), DSM 153 (SEQ ID NO: 5), DYK 138 (SEQ ID NO: 6), DXP4 (SEQ ID NO: 7), EAD 29 (SEQ ID NO: 8), DYC 237 (SEQ ID NO: 9), DUR17 (SEQ ID NO: 10), EAF 39 (SEQ ID NO: 11) and DRW 2 (SEQ ID NO: 12); or b) a polynucleotide complementary to polynucleotide a).   2. Method according to claim 1, characterized in that the isolated polynucleotide comprises a plant promoter selected from the sequences SEQ ID N 1 to 12.   3. Recombinant expression cassette, characterized in that it comprises: a promoter constituted by a polynucleotide sequence as defined in claim 1 or 2; and a heterologous sequence of interest placed under transcriptional control of said promoter and whose expression is sought in the cells of the root of a plant.   4. Expression cassette according to claim 3 characterized in that said sequence of interest is selected from a polynucleotide encoding a protein with herbicidal or antibiotic activity. 20   5. Recombinant vector comprising at least one expression cassette according to one of claims 3 to 4.   6. Host cell transformed with at least one expression cassette according to one of claims 3 or 4 or a recombinant vector according to claim 5.   A transgenic plant or transgenic plant part generated from a cell according to claim 6.   8. Transgenic plant or part of a transgenic plant transformed with at least one expression cassette according to any one of claims 3 to 4.   9. Transgenic plant according to claim 7 or 8 characterized in that it is selected from the following species: Arabidopsis thaliana, rapeseed, tobacco, maize, wheat, barley, sunflower.   10. Process for obtaining a transgenic plant characterized in that it comprises the following steps: a) Obtaining a plant recombinant host cell according to claim 6; b) Regenerating an entire plant from the recombinant host cell obtained in step a); c) Selection of the plants obtained in step b) having integrated the nucleotide sequence of interest placed under the control of the polynucleotide sequence as defined in claim 1.   11. The method of claim 10 characterized in that it comprises the following additional steps: d) crossing between them two transgenic plants as obtained in step c); e) Selection of the plants resulting from the crossing of step d) having retained the transgene.   12. A method for obtaining a transgenic plant characterized in that it comprises the following steps: a) transforming at least one plant cell with a vector according to claim 5; b) Culture of said transformed cells in order to generate a plant containing in its genome an expression cassette as defined according to one of Claims 3 to 4.   13. Transgenic plant obtainable by implementing the method according to any one of claims 10 to 12.   14. Seeds obtained from a transgenic plant according to one of claims 7 to 9 or 13.   A method of protecting a plant from infection with a parasite comprising the following steps: a) transforming the plant with an expression cassette according to any of claims 3 to 4; b) Selection of the plant having integrated the expression cassette.   16. Use of a promoter nucleotide sequence as defined in any one of claims 1 to 4 for expressing a gene in root cells.   17. Use of at least one polynucleotide as defined in one of claims 1 or 2 to express a sequence of interest with herbicidal or antibiotic activity.